The overall goal of our project is to develop advanced technologies based on electrospray ionization with mass spectrometry (MS) and ion mobility spectrometry (IMS) for proteomics and structural biology. The structural determination of protein complexes can play an important role in the fundamental understanding of biochemical pathways. New techniques will be developed to facilitate the characterization of noncovalent protein assemblies. Information related to protein size, protein folding, and protein conformation that can be used to complement information derived from more traditional high resolution structural methods (e.g., x-ray crystallography, nuclear magnetic resonance, cryoelectron microscopy) will be derived from study of the gas phase protein complexes. Modifications to ion mobility spectrometry systems will allow temperature dependences of the ions traveling in the IMS to be used to follow protein folding/unfolding. Experimental and instrumental methods to reduce the charge states of the gas phase complexes, such as gas phase proton transfer reactions, will be developed;cross-sections from their mobility profiles will be determined to study the structural affects of assembly and ligand binding (i.e., conformational differences). Novel developments in intact protein tandem MS will be exploited to define the sites of ligand and protein interactions in a given noncovalent protein complex. Top-down MS/MS will be developed further as a means to determine the specific sites of small molecule ligand binding. The ability to increase the multiple charging of large noncovalent gas phase molecules will be implemented to increase the efficiency of the top-down MS analysis of protein-ligand complexes. The developed methods will be used to determine the binding sites of metals to metalloproteins involved in neurodegenerative disorders and ATP to protein kinases. MS/MS will be used to deliver a "signature" for ATP-binding that can be used to identify new protein kinases. ESI-MS and MS/MS of protein-ligand binding from structurally diverse compound libraries will be developed as a complementary tool for fragment-based ligand design strategies. PUBLIC HEALTH RELEVANCE: We aim to develop more robust analytical technologies that can be applied to provide structural information for biologically important proteins, such as proteins involved in neurodegenerative disorders (e.g., Alzheimer's disease, Parkinson's disease). New methodologies to determine to sites of drug binding to targeted proteins will be developed.